The human immunodeficiency virus (HIV-1) has high mutation rates within certain portions of its genome, permitting rapid evolution of new forms of the the virus that are resistant to drug treatments or can evade the host's immune response. This project investigates the most likely cause for this, inaccurate DNA synthesis by the HIV-1 reverse transcriptase (RT). The RT makes mistakes at a rate sufficient to explain viral variability. Most mistakes made in vitro are initiated by slippage of the template and primer strands, perhaps when the polymerase dissociates and then reassociates with the DNA. In order to gain insight into why the HIV-1 RT is so error-prone for these types of mistakes, we are examining mutant derivatives of the RT, with emphasis on amino acids believed to be important for template-primer interactions. Mutants with alanine substituted for two specific amino acids in alpha helix H of the thumb subdomain that contact the primer strand at positions 3, 4 and 5 nucleotides back from the 3'OH terminus have reduced DNA binding affinity, resistance to AZTTP, reduced processivity and reduced framshift fidelity. These amino acids may make important contacts in the minor groove of the template-primer, a hypothesis that is being examined by determining the effects of additional amino acid substitutions at these key residues. Based on RT structure as determined by X-ray crystallography, studies are also underway to analyze mutants in the I helix and in the "primer grip". Comparative determinations of the fidelity and processivity of HIV-2 and SIV RTs have been completed. Analysis of these wild-type and mutant reverse transcriptases will enhance our understanding of how RTs interact with their substrates and may provide insights into the hypermutability of the AIDS virus. Since these studies focus on mutations resulting from strand slippage, they may also provide insights relevant to several human diseases characterized by mutations that may result from strand slippage during DNA replication. This includes several types of cancer and hereditary diseases.